DNA methylation is a central epigenetic process. All diseases have genetic and/or epigenetic foundations; however only epigenetics can be altered pharmacologically. Thus, epigenetic pathway components are not only excellent epi-therapeutic drug targets, they are also established biomarkers in many acute and chronic diseases. The information stored in 5-methyl-C (mC) is passed down in a heritable and stable fashion; yet, we know that DNA methylation profiles are frequently altered in a number of diseases including Alzheimer?s Disease (AD) where differentially methylated regions (DMRs) have been identified in blood that are similarly found in brain. This means an AD specific pathway or mechanism exists that is mediating alterations in the methylome specifically in peripheral leucocytes, brain and presumably other tissues. Over the past 10y the PI has been working on mC revision pathways and has published numerous papers on the topic. Despite convincing evidence for DMRs in peripheral blood of AD patients, the mechanism of mC re-programming in is unknown. We describe new tools to interrogate mC revisions in neurologically derived cell lines. We devised a screen that uses a neutral GFP gene (i.e., one with no selective pressure) to track changes in DNA methylation in a cellular context. Any forward (more DNA methylation) or backward (less methylation) alters gene expression in a permanent fashion. Cytofluorimetry and cell imaging using engineered reporters, quantifies GFP expression as an indirect readout for mC changes. The method was developed as a screen to identify epi-therapeutics and has been used successfully in this capacity. It is cost effective, simple, robust and will detect biological effectors that alter DNA methylation. We call this the ADEE (AD Epigenetic Effector) screen since it detects epigenetic bio-effectors in AD sera that revise DNA methylation patterns. Also, the ADEE screen works in a lineage specific cell context giving high physiological relevance. Most agree that there is an acute need for peripheral blood biomarkers in AD. We demonstrate that small amounts of serum (5l corresponding to 5%) induced a strong uptick in GFP expression of a heavily methylated reporter gene, revealing the presence of a potent hypomethylating activity in the peripheral blood of AD patients but not in age-match controls. Since ADEEs appear highly active in AD serum, they may represent early tractable biomarkers for early AD onset, disease progression and management. In support of this, epigenome-wide association studies clearly show that changes in DNA methylation are an early antecedent event to clinical manifestation and are tightly associated with AD neuropathology. Archived serum samples (>600) from the GEMS (Gingko biloba Memory Study) will be used to test reliability of the ADEE biomarker. A subset of the patients in the GEM study displayed no cognitive impairment at baseline (at the start of the trial). Over the course of the next 8y in trial, some patients were diagnosed with AD and serum samples were harvested and stored; thus we have sera on the same cohort before and after diagnosis in sufficient numbers to derive high-powered statistical validation regarding positive and negative predictive power of ADEEs. In summary, the ADEE screen is important and well justified for two key reasons: (1) Similar DMRs are found distributed across brain tissue and peripheral blood leukocytes, so we suspected that the ADEE causing (or enabling) the DMRs will be a circulating factor or factors. (2) We developed an innovative cell-based assay proven to test for the presence of ADEE?s. Since we have strong evidence that AD patient serum contains epigenetic effectors, we are uniquely positioned to validate ADEE as a blood biomarker that differentiates AD from normal patients for early detection. We also plan initial characterizations of the ADEEs in order to optimize the test, extend our knowledge of how AD progresses and (as a long term goal) to identify new druggable epigenetic targets. Our specific aims are designed to: 1. Rigorously Test Reliability of ADEE Biomarkers: specificity and selectivity. 2. Optimize, validate and characterize live cell screening operations and assay quality. 3. Carry out validation data analyses in the same patient cohort before and after AD.